Gene Discovery and Heparan Sulfate Biogenesis. Animal cells elaborate a variety of heparan sulfate proteoglycans (HSPGs), which consist of different protein cores and one or more heparan sulfate chains. The HSPGs bind numerous growth factors, cytokines, extracellular matrix components, enzymes and enzyme inhibitors, and they act as receptors or coreceptors for cell signaling, cell attachment, and endocytosis. These interactions depend to a large extent on the composition and the arrangement of sulfated sugars and epimers of uronic acids in the heparan sulfate chains, which in turn depend on the substrate specificity of various biosynthetic enzymes and regulatory factors. Most of the enzymes involved in heparan sulfate biogenesis have been identified, cloned, expressed as recombinant proteins, studied biochemically, and mutated in cells or model organisms. In contrast, a dearth of information exists regarding the mechanisms that give rise to the variable composition and binding properties of heparan sulfate. The central hypothesis of this grant is that genes exist other than those that encode the biosynthetic enzymes, whose expression either modulate the transcription/translation of the enzymes or orchestrate their action to achieve the final composition of heparan sulfate observed in different cell types. Our objective is to search for these regulatory factors through gene- silencing techniques based on stable expression of short hairpin RNAs directed to the whole human genome. Towards this goal, we will (1) adapt high throughput screening assays to identify shRNAs that induce resistance to cytotoxic agents whose action depend on HSPGs (FGF2-Saporin and diphtheria toxin);and (2) identify and characterize genes that modulate the composition of HSPGs by amplification and sequencing the relevant shRNA clones. Genes identified in this way will be sorted into categories based on predicted or known function, and individual candidates will be tested in different cell lines. Detailed structural studies of HS and binding studies with test ligands also will be performed. The significance of this work lies in the potential for discovery of novel regulatory factors involved in HSPG expression. The outcome of these experiments has the wider goal of moving the field into new areas of study. Each factor identified in this way provides a new project for future study and a candidate gene that might explain disorders in which HSPG expression goes awry, such as cancer, inflammation, and atherosclerosis. Insight into the mechanisms that cells use to regulate heparan sulfate composition also might lead to novel drug targets for treating human disease associated with alterations in heparan sulfate formation, such as cancer, inflammation and atherosclerosis. PUBLIC HEALTH RELEVANCE: The significance of this work lies in its potential of uncovering novel regulatory factors involved in heparan sulfate formation. Each factor identified in this way provides new projects for future study. Furthermore, candidate genes might emerge that could explain disorders in which heparan sulfate formation goes awry, such as cancer, inflammation, and atherosclerosis, which in turn could define novel targets for drug development.